Efficient Expression Of Candida Rugosa Lipase CRL1 In Pichia Pastoris And Its Application For Synthesis Of Vitamin E Acetate

Candida rugosa lipase(CRL)is considered to be a versatile and promising biocatalyst,widely used in the pharmaceutical,food,cosmetics and energy industries.Commercial preparations of CRL,comprising 8 kinds of isoenzymes,are mainly produced by the wild-type strains of ATCC 14830 and Type VII,in which CRL1 has the highest expression level and lipase activity.Due to the distinction in composition and proportion of isoenzymes,there is a significant difference in substrate selectivity and catalytic efficiency between commercial enzymes.Therefore,it is important to isolate and produce single Candida rugosa lisoenzyme,especially the CRL1,which is meaning for further research.Pichia pastoris is the most widely used heterogenous protein expression system,which is benefical to the efficient expression and industrial production of heterogenous proteins.Pichia pastoris surface display technology is a cost-effective immobilization method for enzymes,which can improve the stability and reusability of enzymes,resulting in a lower production cost.In this study,Pichia pastoris was used as a host cell to construct secretory and cell surface display strains of CRL1 to produce the lipase efficiently.On this basis,the enzymological properties of both two types of CRL1 were studied,and then a green process for the synthesis of vitamin E acetate in a solvent-free system using CRL1 as biocatalyst was established and optimized,laying a foundation for its large-scale industrial application.The main research work and innovations of this article are as follows:(1)The optimized gene encoding Candida rugosa lipase CRL1 was synthesized in vitro,cloned into the vector p HKA and expressed in Pichia pastoris,producing a lipase activity of 39.73 U/m L towards p-nitrophenyl butyrate(pNPB)after induction for 120 h in flask fermentation.Modification of AOX1 promoter and ?-factor signal peptide together increased the lipase activity by 60.18%,achieving 63.63 U/mL.Then the CRL1 product was purified 5.41-fold(984.52 U/mg)with a 33.8% yield after ultrafiltration,ammonium sulphate precipitation and anion exchange chromatography.The recombined CRL1 was characterized by the optimal temperature and pH at 40? and 7.5,respectively.Besides,it showed a poor thermal stability and pH activity.In 3-L fed-batch fermentation,the lipase activity achieved 910 U/mL,which is 13 folds higher than the flask fermentation level.(2)Candida rugosa lipase CRL1 was successfuly displayed on the cell wall of Pichia pastoris using the modified FS anchor system for the first time.Lipase activity of the recombinant strain GS115/pKFSCRL1 achieved 7500 U/g in shake flask culture,while higher activity of 19,800 U/g(increased by 164%)was obtained by a five copy construct overexpressing HAC1 factor,namely GS115/pKZA-5FSCRL1-HAC1.Besides,the results of flow cytometry analysis had demonstrated that CRL1 was successively displayed on the cell surface of Pichia pastoris,and the further quantitative analysis showed a more than 90% displaying rate.Displayd CRL1 was characterized by the optimal temperature and pH at 50? and 8.5,respectively.Moreover,it had a better thermostability and pH stability than free lipase.Displayed-CRL1 also showed remarkable stability in organic solvents,and different kinds of metal ions and surfactants were investigated their influence on the lipase activity.In 3-L fed-batch fermentation,the lipase activity of the displayed CRL1 achieved 18,800 U/g,similar wth the flask fermentation level.(3)An efficient green process to synthesize VEA in solvent-free system using the recombinant CRL1 as biocatalyst was developed and optimized.The optimum conditions were: 200 mg d-?-tocopherol,1.0 m L acetic anhydride,100 mg CRL1,at 60? and 200 rpm.Under these conditions,the yields of VEA achieved above 97.00% after 9 h.Furthermore,the reaction mixture was neutralized with sodium carbonate,filtered by suction filtration,washed with n-hexane and evaporated under reduced pressure.Consequently,the final product was obtained with a purity of 96.5% of VEA,and then it was characterized and confirmed by FTIR and GC-MS spectra further.